CIRCULATING TUMOR CELLS

A typical cancerous tumor contains millions or even billions of cells harboring genetic mutations driving them to grow, divide, and invade the local tissue in which they’re embedded. However, as the cells proliferate, they don’t all stay in the neighborhood. Some cells slough off the edges of a tumor and are swept away by the bloodstream or lymphatic system. These so-called circulating tumor cells (CTCs) can remain loose in circulation, cluster together as they travel, or lodge themselves in new tissues. Whatever their path, their common origin means that CTCs hold information about a tumor, information that researchers think could be key to cancer diagnosis or treatment.

Cancer patients have only between 5 and 50 CTCs per teaspoon of blood, so their presence is dwarfed by blood cells. However, in the past decade emerging technologies have, for the first time, allowed the isolation of CTCs from patients’ blood samples. Some methods, among the first established, rely on the cells’ physical properties. When a blood sample settles or is spun in a centrifuge, red blood cells, white blood cells, and other components of blood separate into layers. Based on their buoyancy, CTCs can be found in the white blood cell fraction. Then, because CTCs are generally larger than white blood cells, a size-based filter can divide the cell types.

SOME FACTS:

Everyone with cancer has CTCs!

CTCs are NOT yet rapidly replicating

Therefore CTCs are NOT killed by chemotherapy or surgery

CTCs are ONLY kept dormant by a healthy immune system

Chemotherapy and radiation greatly inhibit the immune system

Therefore Chemotherapy and radiation greatly increase metastatic risk

CTCs Discovery

In 1869, pathologist Thomas Ashworth noticed some unusual cells in the blood of a patient who had died of cancer. The cells didn’t look like normal blood cells; instead, they were similar in appearance to those found in the numerous solid tumors present all over the patient’s body. Ashworth speculated that perhaps the cells were derived from the existing tumors, and could help explain the distribution of the patient’s multiple lesions (Australian Med J, 14:146-49, 1869).

Scientists now believe that these so-called circulating tumor cells (CTCs) play a key role in metastasis. Because CTCs can be obtained through routine blood draws—a procedure that is much easier and less invasive than a tumor biopsy, making it amenable to repetition—many scientists are hopeful that the cells could be used to detect cancer and metastases at an early stage. CTCs could also help doctors plot the molecular signature of an individual’s tumor over time, monitor a tumor’s responsiveness to therapy, and identify targets for the development of personalized therapies.

Why they are IGNORED by standard Oncology

However, pharmaceutical research is driven by money. Since the main medical cancer therapies (chemotherapy, radiation and surgery) do NOT kill CTCs, the chance of cancer metastasis following chemo, radiation and surgery greatly increases!

Let’s think about it – standard oncology makes their money selling therapies that may kill cancer cells but actually protect circulating tumor cells, setting the patient up for cancer to “come back with a vengeance” within a short period of time. Addressing CTCs may greatly disrupt money flow from standard therapies!

A recent University of Michigan study stated that, “This study confirms the prognostic significance of CTCs in patients with MBC receiving first-line chemotherapy. For patients with persistently increased CTCs after 21 days of first-line chemotherapy, early switching to an alternate cytotoxic therapy was not effective in prolonging OS. For this population, there is a need for more effective treatment than standard chemotherapy.” They suggest that for many cancer patients, there is even an INCREASE in CTCs following chemotherapy! For these the study goes on to suggest that there may be a “need for more effective treatment than standard chemotherapy.”

CTCs and their role in malignant disease:

Circulating Tumour Cells (CTCs) sparked scientific interest over fifty years ago and their detection and analysis is proving to be an invaluable tool in the individualisation of cancer diagnosis and treatment [3].

It is very well established that Circulating Tumour Cells are absolutely essential for the establishment of metastases: they function as the single haematological route of malignant neoplasias and metastases cannot occur without them [1]. In fact, ‘metastatic insufficiency’ is officially defined as the elimination of CTCs [4]. Regardless of their critical role in the metastatic cascade and despite the need for their detection and analysis as a widespread tool used in cancer management [5,6,7], a definition of CTCs has yet to enter a medical dictionary.

CTCs are a subpopulation of tumour cells derived from the primary cancer site that have:

Detached from the primary tumour mass [8]

Adopted genetic mutations that enabled migration through the basement membrane (if the tumour is of epithelial origin) and the extracellular matrix [4,9]

Entered into the peripheral blood stream where they circulate as tumour cells with metastatic potential – this is the point at which they are termed ‘Circulating Tumour Cells.’ [1,10]

Have the potential to disseminate and proliferate as a metastatic lesion [1,4]

Can stimulate angiogenesis [1,10]

Have stem-cell like properties (see below) [1,2,11]

Survival of CTCs in the circulation requires evasion of anoikis and of the immune system. There are complex mechanisms present in CTCs that allow for this prevarication to occur [9,12,13]. When the intercellular signalling is appropriate, CTCs extravase from the circulation, disseminate in a tissue foreign to that of the primary lesion, and proliferate in the ‘permissive’ organ [1,3,5,10,14]. This proliferating mass forms a secondary cancer at a site foreign to that of the primary cancer [1].

STEPHEN PAGET’S ‘SEED AND SOIL’ HYPOTHESIS

Stephen Paget’s well-recognised ‘seed and soil’ hypothesis states that metastases exhibit tropism, i.e. the organ site wherein they disseminate and form a secondary tumour is not random [5,14]. The organ site of a metastasis is the ‘soil’ which is absolutely biologically the ideal place for a specific ‘seed’ (CTC) to grow [1]. Both the CTC (seed) and the organ site (soil), which will harbour the metastases, have biomarkers that specifically recognise and interact with each other [1]. Together they facilitate the development of the environment necessary for a metastatic lesion to develop and thrive. [1,3,5,10,14] CTCs have adopted genetic mutations that equip them to respond to local growth factors and stimulate neovascularisation in the microenvironment of the new site. [1,4,5,14]. These biological markers on CTCs may differ entirely from the markers of the bulk of primary cancer cells [1,11].

HETEROGENEITY OF TUMOUR CELLS

Understanding the heterogeneous nature of tumour cells is necessary in order to fully appreciate the critical role CTCs play in the formation of metastases [15]. A vast number of the CTC characteristics are yet to be determined, however, it is known that CTCs are likely to have heterogeneous biomarkers to that of the parent tissue and other subpopulations of the primary tumour [1,16]. Common CTC properties that identify them as heterogeneous to other primary cancer cells are their increased invasiveness, their heightened resistance to threat, and their biological likeness to stem or progenitor cells [1,4,6].

The heterogeneous nature of tumour has the following consequences:

Classification and morphological analysis of tumour cells from a surgical biopsy may differ to the character of the tumour’s CTCs [1,11].

The majority of the cells of the biopsy will not have initiating capacity and therefore may be less relevant in terms of diagnosis and treatment [1].

CTCs have the potential to behave totally differently to the original primary cancer cells and respond to entirely different treatments [1,11].

CTCs and Tumour Initiating Cells (TICs)

There is confusing terminology existing in the literature about tumour stem cells. Tumour cells that have progenitor/stem cell characteristics and are responsible for tumour progression are called Tumour Initiating Cells (TICs) [15]. They are known colloquially as ‘Cancer Stem Cells’ (CSCs) [17]

CTCs share similar genotypic and phenotypic characteristics with Tumour Initiating Cells (TICs) [1,6]. CTCs have the capacity to self-renew, to divide asymmetrically, for genetic adaptation, and to accumulate mutations [4]. They have the ability to sustain tumour genesis and growth, and to initiate tumours with multiple descendent lines. [2,11,18]. CTCs may circulate as non-proliferating tumour cells, potentiating their resistance to chemotherapy [19,20]. They can transition from this non-proliferating pluripotent-progenitor cell phenotype into a proliferating cell upon dissemination [21].

The similarities that CTCs have to cancer stem cells may explain the eventual relapse of disease in a patient previously considered to be in remission following primary therapy [6,15,22].

The sub-population of neoplastic cells that have stem cell properties are known to:

be responsible for tumour progression [15]

have unique biomarkers that may correspond to radio- and chemotherapy resistant mechanisms [23]

be derived from and regulated by both genetic and epigenetic programs [24]

If therapy is to be targeted toward cells responsible for tumour progression, these epigenetic determinants of mutations need to be considered [24].

Cancer treatments may be unsuccessful if they fail to target the specific minority subpopulation of tumour cells that have capacity for invasion and tumour initiation [15]. These populations are an absolutely essential target for therapy and if metastatic disease is to be prevented. [15,25]

What can CTCs tell us about the patient’s malignancy?

“CTCs have a wealth of clinical information in the evaluation of tumour progression, prediction of long term prognosis, identification of patients who are likely to respond to treatment of curative intent, and assessment of likelihood of recurrence.” [4]

The identification and analysis of CTCs is emerging as an essential clinical tool in the diagnosis of malignancy, and in the monitoring of disease progression and effect of cancer treatment [1,3,26,27].

CTC detection and analysis is a valuable tool in the management of cancer because it enables the following information to be realised:

EVALUATION OF TUMOUR PROGRESSION IN REAL-TIME

Analysis of CTCs enriched from the peripheral blood of patients with advanced or metastasising cancer represents the real-time biopsy that has been up until this point impossible without surgical intervention [11,18]. Detection of CTCs in the peripheral circulation of cancer patients indicates the presence of metastatic disease [1,4,11]. Due to the ease of sample collection, it is possible to monitor tumour progression and stage, and assist in determining the success of cancer treatment [5]. CTC count in the peripheral blood of a patient is indicative of tumour stage, tumour progression, and success of treatment [1]. The difference in CTC count between two samples, taken prior to and following surgery or cancer therapy, can inform the practitioner of the success of the treatment [5]. CTC count falls significantly with the regressing of disease, and similarly CTC count rises with the advancement of the malignancy [4,5].

The CTC count is indicative of tumour stage [3]. The numerical value which determines how advanced the cancer is will differ across the various types of malignant neoplasias, and their comparative averages have already been determined [4]. For example, more than 5 CTCs per 7.5ml of peripheral blood of patients with breast cancer is considered to be a progressive disease.

PREDICTION OF LONG-TERM PROGNOSIS

The presence of CTCs in the peripheral circulation has been confirmed as an independent prognostic indicator [1,5]. CTC detection is predictive of clinical outcome and overall survival rate in multiple malignancies [1,5]. The prognostic significance of CTCs relates to time to disease progression and prediction of recurrence, even after therapy of curative intent [1,4,14].

CTC detection in the blood may override the standard prognostic indicators [2,4]. Specifically, detection and analysis of CTCs may be a more accurate predictor of clinical outcome in terms of Overall Survival than standard prognostic indicators [2]. Multivariate analysis has shown that CTC count is an independent prognostic indicator irrespective of all other variables [1,5,6].

Due to the similarity between Cancer Stem Cells and CTCs, (i.e. their characteristics of longevity, capacity for tumour initiation, self renewal and proliferative ability), the presence of CTCs at the time of diagnosis and treatment, may explain the eventual relapse of disease in patients who have previously been ‘in remission’ after primary therapy [6].

IDENTIFICATION OF PATIENTS WHO ARE LIKELY TO RESPOND TO TREATMENT OF CURATIVE INTENT

It is difficult to predict the biological fate of the cancer from biopsies obtained from the primary cancer [28]. A significant number of patients experience metastatic disease following primary therapy due to the treatment’s inability to target the more aggressive metastasising population [3,29] and the metastatic inducing nature of biopsies.

The biological fate of malignancies is determinable through the detection and bio-characterisation of CTCs [18,28].

ASSESSMENT OF LIKELIHOOD OF RECURRENCE

CTC detection and analysis makes it possible to assess the risk of disease recurrence after therapy of curative intent [1,4]. CTC count in the peripheral circulation before both surgery and chemotherapy or other treatment is the marker that can independently predict the early recurrence in patients with cancer [14]. Novel enrichment and molecular analytic techniques have made it possible to detect metastasising disease that is undetectable using conventional imaging techniques [4].

The detection and isolation of CTCs

Circulating Tumour Cells (CTCs) are events in the peripheral circulation of cancer patients with malignant disease [7]. They can be reliably detected, isolated, cultured and analysed using immunocytochemical and biomolecular techniques [1,2,7,16].

When used in isolation, each of the available detection methods have their advantages and pitfalls [1]. There is yet to be a standardised CTC- enrichment technique, however, the FDA has approved the Veridex™ (Johnson & Johnson) CellSearch® device for CTC detection in late stage breast cancer, colon cancer and prostate cancer patients only [1,4]. CellSearch® is known for its high specificity, but poor sensitivity [4]. Numerous studies indicate that using a combination of the more recent physical and immunochemical techniques overcomes the disadvantages each method may have when used on their own [1,2,6,7,16].

CTCs either express or, given certain conditions, have the potential to express renegade proteins that are associated with the robustness of malignant tumours [31]. The earlier that cells with tumour initiation capacity are detected and analysed, the sooner an individualised treatment design is possible. [15,32] The identification of both surface and intracellular markers that indicate metastatic progression are they key to detecting the CTCs in the blood.

As yet it is too complex to detect and isolate tumour stem-cells within a tumour mass due to the lack of identifiable stem cell markers [15]. The detection of CTCs however overcomes this problem: their significance lies in their similarity to tumour stem cells, and they are easily isolated from the peripheral circulation [1,3].

Methods of enrichment and gene profiling may involve one or a combination of the following:

PHYSICAL:

Centrifugation: isolating CTCs based on their gradient-density [1,4,16]

• Reverse Transcriptidase Polymerase Chain Reaction detects genetic mutations in the DNA of CTCs. Primers or probes are designed which base-pair with the specific gene or chromosomal sequence (mutation) of interest, thereby identifying their presence. Multiple sequences (in fact the entire genomic sequence) can be analysed simultaneously [1].

DNA MICROARRAY:

• DNA microarrays enables the identification of genes, determines the active expression of genomic sequences, and detects oncogenic mutations/polymorphisms present in the nucleic acids of any cell [33]. The process makes biochemical calculations of the mRNA that is expressed in cells, hence revealing the cell’s molecular biology. DNA-microarrays can analyse multiple genes simultaneously and have revolutionary diagnostic potential [32,34].

FLOW CYTOMETRY:

• Flow Cytomertry examines the biomolecular footprint of cells. In a nutshell, a cell is tagged for specific constituents and exposed to a laser beam of light. The presence of proteins and sub-cellular molecules in/on the cell will cause the light to fragment in a pattern that, in turn, identifies their existence. The patterns created by the scattering of light can be detected and analysed [35,36,37,38,39].

The significance of CTCs in terms of diagnosis and treatment

“It is undeniable that CTCs have enormous research potential for individualised medicine in the future.” [3]

Molecular diagnostics hold great promise for individualised diagnosis of cancer [15]. It is possible not only to detect defunct proteins that regulate the cell cycle but also to scan the entire genome of metastasising cells and detect the genes associated with cancer progression prior to them even being transcribed or expressed [2,30]. Molecular technology also allows the detection and testing of resistant or chemosensitive mechanisms existing or dormant within the tumour cell [40]. Such knowledge of the biology of a patient’s cancer allows clinicians to select effective targeted therapies, to monitor the effects of treatment in real-time, and to adapt treatment according to new mutations or protein expression that may have arisen [2,30]. Detection of these mechanisms is highly valuable in effective cancer management [3,18].

A major factor contributing to the possibility of individualised diagnosis through detection and analysis of CTCs is simply the ease of sample collection and accessibility of the cells [3]. Traditionally, clinicians have had to obtain a tissue sample that needs preservation in formalin and fixing in paraffin in order to analyse cancer cells [1]. Analysis of cancer cells isolated from the peripheral circulation overcomes this hassle as well as providing a continuous source of DNA, being free of selection bias, being instantaneous, less expensive and far less invasive than a biopsy surgically removed from a solid tumour [4,15].

Individualised treatment arises from the possibility of assessing treatment efficacy, and monitoring the changing molecular biology of heterogeneous subpopulations of cancer cells [41]. Heterogeneous mutations and protein expression can be detected through highly sensitive methods of analysis of CTCs, deeming CTCs potentially central to the tailoring of cancer therapy [3,4].